Eunsook Chung

Molecular cloning and characterization of the soybean DEAD-box RNA helicase gene induced by low temperature and high salinity stress.

A novel gene encoding a DEAD-box RNA helicase designated as GmRH was isolated from soybean. Amino acid sequence alignment and phylogenetic tree analysis revealed a close relationship between GmRH and other orthologous DEAD-box RNA helicases from other plant species. Structural motif analysis revealed that the bipartite lysine rich nuclear localization signal (NLS) is present in the N-terminal variable region of GmRH and that there are ten conserved motifs found in DEAD-box RNA helicase proteins. Southern blot analysis revealed the presence of 2 copies of GmRH in the soybean genome. Northern blot analysis demonstrated that the RNA expression of the GmRH was induced during low temperature or high salinity stress, but not by the exogenous application of abscisic acid or drought stress. Subcellular localization studies showed that GmRH((1-355))-GFP is localized in the nucleus, whereas GmRH((130-355))-GFP is localized both in the cytoplasm and in the nucleus. This provides the evidence that the N-terminal region predicted as NLS is essential for nuclear targeting of the GmRH protein in the plant cell. Purified GST-GmRH recombinant protein was shown to unwind dsRNA independent of ATP in vitro. Here, we propose that GmRH plays an important role in RNA processing during low temperature and high salinity stresses in plants.

Genome-wide analysis and molecular characterization of heat shock transcription factor family in Glycine max.

Heat shock transcription factors (Hsfs) play an essential role on the increased tolerance against heat stress by regulating the expression of heat-responsive genes. In this study, a genome-wide analysis was performed to identify all of the soybean (Glycine max) GmHsf genes based on the latest soybean genome sequence. Chromosomal location, protein domain, motif organization, and phylogenetic relationships of 26 non-redundant GmHsf genes were analyzed compared with AtHsfs (Arabidopsis thaliana Hsfs). According to their structural features, the predicted members were divided into the previously defined classes A-C, as described for AtHsfs. Transcript levels and subcellular localization of five GmHsfs responsive to abiotic stresses were analyzed by real-time RT-PCR. These results provide a fundamental clue for understanding the complexity of the soybean GmHsf gene family and cloning the functional genes in future studies.

Overexpression of VrUBC1, a Mung Bean E2 Ubiquitin-Conjugating Enzyme, Enhances Osmotic Stress Tolerance in Arabidopsis

The ubiquitin conjugating enzyme E2 (UBC E2) mediates selective ubiquitination, acting with E1 and E3 enzymes to designate specific proteins for subsequent degradation. In the present study, we characterized the function of the mung bean VrUBC1 gene (Vigna radiata UBC 1). RNA gel-blot analysis showed that VrUBC1 mRNA expression was induced by either dehydration, high salinity or by the exogenous abscisic acid (ABA), but not by low temperature or wounding. Biochemical studies of VrUBC1 recombinant protein and complementation of yeast ubc4/5 by VrUBC1 revealed that VrUBC1 encodes a functional UBC E2. To understand the function of this gene in development and plant responses to osmotic stresses, we overexpressed VrUBC1 in Arabidopsis (Arabidopsis thaliana). The VrUBC1-overexpressing plants displayed highly sensitive responses to ABA and osmotic stress during germination, enhanced ABA- or salt-induced stomatal closing, and increased drought stress tolerance. The expression levels of a number of key ABA signaling genes were increased in VrUBC1-overexpressing plants compared to the wild-type plants. Yeast two-hybrid and bimolecular fluorescence complementation demonstrated that VrUBC1 interacts with AtVBP1 (A. thaliana VrUBC1 Binding Partner 1), a C3HC4-type RING E3 ligase. Overall, these results demonstrate that VrUBC1 plays a positive role in osmotic stress tolerance through transcriptional regulation of ABA-related genes and possibly through interaction with a novel RING E3 ligase.

Accumulation of anthocyanin and associated gene expression in radish sprouts exposed to light and methyl jasmonate.

Radish (Raphanus sativus) sprouts have received attention as an important dietary vegetable in Asian countries. The flavonoid pathway leading to anthocyanin biosynthesis in radishes is induced by multiple regulatory genes as well as various developmental and environmental factors. This study investigated anthocyanin accumulation and the transcript level of associated genes in radish sprouts exposed to light and methyl jasmonate (MeJA). The anthocyanin content of sprouts exposed to light and treated with MeJA was higher than that of sprouts grown under dark conditions without MeJA, and the highest anthocyanin content was observed within 6-9 days after sowing (DAS). Transcript levels of almost all genes were increased in radish sprouts grown in light conditions with 100 μM MeJA relative to sprouts grown under dark conditions with or without MeJA treatment, especially at 3 DAS. The results suggest that light and MeJA treatment applied together during radish seedling development enhance anthocyanin accumulation.

Fermented Viola mandshurica Inhibits Melanogenesis in B16 Melanoma Cells

We assessed the effects of chloroform extract of fermented Viola mandshurica (CEFV) on melanogenesis B16 melanoma cells. CEFV treatment significantly decreased melanin content and tyrosinase activity in dose-dependent manners. To elucidate the mechanism of the inhibitory effects of CEFV on melanogenesis, we performed RT-PCR and Western blotting for melanogenesis-related genes such as tyrosinase, tyrosinase-related protein-1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor (MITF). CEFV strongly inhibited mRNA as well as the protein expression of tyrosinase and MITF, but had no significant effect on TRP-1 or TRP-2 expressions. It markedly decreased the phosphorylation of cAMP responsive element binding protein (CREB), and induced the duration of extracellular signal-regulated kinase (ERK) activation, leading to reduction of MITF expression and subsequently that of tyrosinase. Therefore, we suggest that CEFV induces downregulation of melanogenesis through decreased CREB phosphorylation and ERK activation.

Altered Gene Expression and Intracellular Changes of the Viable But Nonculturable State in Ralstonia solanacearum by Copper Treatment

Environmental stresses induce several plant pathogenic bacteria into a viable but nonculturable (VBNC) state, but the basis for VBNC is largely uncharacterized. We investigated the physiology and morphology ofthe copper-induced VBNC state in the plant pathogen Ralstonia solanacearum in liquid microcosm. Supplementation of 200 μM copper sulfate to the liquid microcosm completely suppressed bacterial colony formation on culture media; however, LIVE/DEAD BacLight bacterial viability staining showed that the bacterial cells maintained viability, and that the viable cells contain higher level of DNA. Based on electron microscopic observations, the bacterial cells in the VBNC state were unchanged in size, but heavily aggregated and surrounded by an unknown extracellular material. Cellular ribosome contents, however, were less, resulting in a reduction of the total RNA in VBNC cells. Proteome comparison and reverse transcription PCR analysis showed that the Dps protein production was up-regulated at the transcriptional level and that 2 catalases/peroxidases were present at lower level in VBNC cells. Cell aggregation and elevated levels of Dps protein are typical oxidative stress responses. H2O2 levels also increased in VBNC cells, which could result if catalase/peroxidase levels are reduced. Some of phenotypic changes in VBNC cells of R. solanacearum could be an oxidative stress response due to H2O2 accumulation. This report is the first of the distinct phenotypic changes in cells of R. solanacearum in the VBNC state.

Molecular Cloning and Characterization of Soybean Cinnamoyl CoA Reductase Induced by Abiotic Stresses

National Academy of Agricultural Science, RDA, Suwon 441-707, Korea (Received on August 10, 2010; Accepted on October 26, 2010)Suppression subtractive hybridization was used toisolate wound-induced genes from soybean. One of thewound-induced genes, gmwi143 designated as GmCCR,showed high homology with genes encoding cinnamoyl-CoA reductase (CCR; EC 1.2.1.44). Deduced aminoacid sequences encoded by GmCCR showed the highestidentity (77%) with those of Acacia CCR. There are 2CCR genes highly homologous to GmCCR in soybeangenome based on Phytozome DB analysis. RNA expre-ssion of GmCCR was specifically induced by local andsystemic wounding, drought, high salinity or by ultra-violet stress. Our study suggests that GmCCR may beinvolved in resistance mechanism during abiotic stressesin plants. Keywords :cinnamoyl-coA reductase, lignin, soybean, wound-ing, UVLignin, a major component of secondary cell walls, isdeposited mainly in the vascular tissues during plantdevelopment and environmental signals (Boerjan et al.,2003). Lignins contribute to major functions in plants, suchas stem rigidity, water transport in xylem and are alsoinvolved in defense reactions against wounding, predatorsor pathogens (Vance et al., 1980). It has been reported thatlignin biosynthesis is regulated by developmental signal inthe vascular tissues and by defense responses in plant(Lauvergeat et al., 2001).The biosynthesis of lignins begins with the commonphenylpropanoid pathway, starting from phenylalanine andleading to cinnamoyl CoAs which are the common pre-cursors of a wide range of phenolic compounds. These CoAesters subsequently are changed to monolignols via tworeductive steps catalyzed by cinnamoyl CoA:NADP oxido-reductase (CCR, EC 1.2.1.44) and cinnamyl alcohol de-hydrogenase (CAD, EC 1.1.1.195). Lignins result from theoxidative polymerization of monolignols. Several genes encoding CCR have been shown to betranscriptionally induced by developing xylem tissues or bybiotic stress (Kim et al., 2000; Lacombe et al., 1997;Lauvergeat et al., 2001). Lacombe et al. (1997) showed thatEucalyptus CCR transcript was expressed in lignified organssuch as leaves, stems and roots. By in situ hybridizationtechnique, EuCCR was strongly expressed in the differ-entiating xylem zone but was not present in the secondaryphloem fibers or in the young periderm (Lacombe et al.,1997). Rice OsCCR1 RNA expression was induced insuspension cell cultures treated with sphingolipid elicitorpurified from rice blast Magnaporthe grisea (Kawasaki etal., 2006). Previously, soybean chip analysis showed thatRNA expression of CCR invovled in phenylpropanoidpathway were induced in the syncytia laser microdissectedsoybean roots infected with soybean cyst nematode (Klinket al., 2009).There are 11 putative CCR homologues sharing homo-logy identities raging from 82.8 to 32.8% in Arabidopsisgenome (Costa et al., 2003). Arabidopsis AtCCR1 transcriptwas expressed in the stem and floral tissues (Lauvergeat etal., 2001) and the irregular xylem4 mutant (irx4) defectivein the AtCCR1 gene showed severely reduced lignin con-tents (50%) compared to the wild type (Jones et al., 2001).Lauvergeat et al. (2001) suggested that AtCCR2 responsiveto pathogen infection of Xanthomonas campestris pv.campestris may play a role in the formation of phenoliccompounds associated with HR in Arabidopsis (Lauvergeatet al., 2001). In the present study, we have isolated the full-lengthcDNA of GmCCR encoding CCR-like protein regulated byabiotic stresses. We propose that the GmCCR is involved indefense responses during environmental stresses in plants.

Hyul-Tong-Ryung suppresses PMA-induced MMP-9 expression by inhibiting AP-1-mediated gene expression via ERK1/2 signaling pathway in MCF-7 human breast cancer cells.

Our previous study has demonstrated that the methanol extract of Hyul-Tong-Ryung (HM) specifically suppresses the phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) production through the inhibition of MMP-9 mRNA expression in MCF-7 human breast carcinoma cells. However, the molecular mechanisms involved in transcriptional suppression of MMP-9 by HM in PMA-induced MCF-7 cells are not known. In this study, we aimed to elucidate the molecular mechanisms involved in the inhibition of MMP-9 expression by HM in PMA-induced MCF-7 cells. The results of promoter assay and EMSA showed that HM specifically inhibits MMP-9 gene expression by blocking PMA-stimulated activation of activator protein-1 (AP-1). In addition, PMA-stimulated phosphorylation of extracellular signal regulated kinase1/2 (ERK1/2) was suppressed by HM treatment, whereas the phosphorylation of either c-Jun N-terminal kinase (JNK) or p38 mitogen-activated protein kinase (MAPK) was not affected. HM could inhibit the PMA-induced MMP-9 expression through suppression of the transcriptional activity of MMP-9 gene in MCF-7 cells. These results indicate that HM inhibits PMA-induced MMP-9 expression by blocking the activation of activator protein-1 (AP-1) via extracellular signal regulated kinase1/2 (ERK 1/2) signaling pathway.

Characterization of a Soil Metagenome-Derived Gene Encoding Wax Ester Synthase.

A soil metagenome contains the genomes of all microbes included in a soil sample, including those that cannot be cultured. In this study, soil metagenome libraries were searched for microbial genes exhibiting lipolytic activity and those involved in potential lipid metabolism that could yield valuable products in microorganisms. One of the subclones derived from the original fosmid clone, pELP120, was selected for further analysis. A subclone spanning a 3.3 kb DNA fragment was found to encode for lipase/esterase and contained an additional partial open reading frame encoding a wax ester synthase (WES) motif. Consequently, both pELP120 and the full length of the gene potentially encoding WES were sequenced. To determine if the wes gene encoded a functioning WES protein that produced wax esters, gas chromatography-mass spectroscopy was conducted using ethyl acetate extract from an Escherichia coli strain that expressed the wes gene and was grown with hexadecanol. The ethyl acetate extract from this E. coli strain did indeed produce wax ester compounds of various carbon-chain lengths. DNA sequence analysis of the full-length gene revealed that the gene cluster may be derived from a member of Proteobacteria, whereas the clone does not contain any clear phylogenetic markers. These results suggest that the wes gene discovered in this study encodes a functional protein in E. coli and produces wax esters through a heterologous expression system.

Purification and structure determination of gelatinase and collagenase inhibitors from Viola patrinii fermentation extracts.

Investigation of collagenase and gelatinase inhibitory natural components afforded two isoflavonoids. Two isoflavonoids, tectorigenin-7-O-β-D-glucoside (1) and luteolin-7-O-β-D-glucuronopyranoside (2), were isolated from ethyl acetate fraction of Viola patrinii fermentation extracts (VPFE). Of these, compounds 1 and 2 exhibited collagenase inhibitory activity (IC50) at a concentration of less than 1.5 μM, and compound 2 showed gelatinases A and B inhibitory activity (IC50) at 0.3 μM and 0.8 μM, respectively.